1. Field of the Invention
This invention relates to novel multibinding compounds (agents) that inhibit the enzyme cyclooxygenase-2 (COX-2) and to pharmaceutical compositions comprising such compounds. Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of various disorders mediated by COX-2, such as inflammation, pain, fever and the like.
2. References
The following publications are cited in this application as superscript numbers:
1 W. L. Smith et al., Adv. Immunol. 1996, 62, 167-215.
2 D. Picot et al., Nature 1994, 367, 243-249.
3 C. Luong et al., Nat. Struct. Biol. 1996, 3, 927-933.
4 R. G. Kurumbail et al., Nature 1996, 384, 644-688.
5 J. K. Gierse et al, J. Biol. Chem. 1996, 271, 15810-15814.
6 E. Wong et al., J. Biol. Chem. 1997, 272, 9280-9286.
7 P. Prasit et al., Annual Reports in Medicinal Chemistry 1997, 32, 211-220.
8 J. Med. Chem. 1997, 40, 1619-1633; 1634-1647.
All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid to prostaglandin H2, the first committed step in the biosynthesis of prostaglandins, such as prostacyclin and thromoxanes.1 It has been known for some time that the enzyme COX is the target of certain non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, naproxen, indomethacin and the like. More recently, it has been discovered that there are two isoforms of COX, designated COX-1 and COX-2.
The COX-1 isozyme is constitutively expressed in many tissue types where it produces relatively small amounts of prostaglandins necessary for maintaining organ and tissue homeostasis. In constrast, COX-2 is transiently expressed in a limited number of cell types, including synovial cells, fibroblasts, macrophages and monocytes. In such cells, high level expression of COX-2 is rapidly induced in response to certain inflammatory agents, hormones, growth factors, cytokines and the like, resulting in various disorders such as inflammation, pain, fever and the like.
Unfortunately, most currently-marketed NSAIDs are typically equipotent inhibitors of both isozymes of COX. Since such drugs inhibit COX-1 as well as COX-2, they interfere with various prostaglandin-regulated processes not associated with the inflammation process and other related disorders. As a result, many NSAIDs cause severe side effects, such as stomach ulcers and renal damage, which limit their effectiveness as therapeutics. Accordingly, selective inhibitors of COX-2 would have significant advantages over currently-marketed NSAIDs.
It has now been discovered that selective COX-2 inhibitors having surprising and unexpected properties can be prepared by linking from 2 to 10 ligands to one or more linkers, wherein each ligand is a moiety capable of binding to COX-2. Such multibinding compounds provide improved biological and/or therapeutic effects compared to the aggregate of the unlinked ligands due to their multibinding properties.
This invention is directed to novel multibinding compounds (agents) that inhibit cyclooxygenase-2 (COX-2). The multibinding compounds of this invention are useful in the treatment and prevention of disorders mediated by COX-2, such as inflammation, pain, fever and the like.
Accordingly, in one of its composition aspects, this invention provides a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding to cyclooxygenase-2; and pharmaceutically-acceptable salts thereof.
In another of its composition aspects, this invention provides a multibinding compound of formula I:
(L)p(X)qxe2x80x83xe2x80x83I
wherein each L is independently a ligand comprising a moiety capable of binding to cyclooxygenase-2; each X is independently a linker; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; and pharmaceutically-acceptable salts thereof.
Preferably, q is less than p in the multibinding compounds of this invention.
Preferably, each ligand, L, in the multibinding compound of formula I is independently selected from a compound of formula IA, IB or IC: 
wherein
ring A, together with the atoms to which it is attached, forms a 4, 5 or 6-membered carbocyclic or heterocyclic ring selected from the group consisting of aryl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic;
ring B, together with the atoms to which it is attached, forms a 4, 5 or 6-membered heterocyclic ring selected from the group consisting of heteroaryl and heterocyclic;
ring C, together with the atoms to which it is attached, forms a 4, 5 or 6-membered heterocyclic ring selected from the group consisting of heteroaryl and heterocyclic;
R1 is selected from the group consisting of xe2x80x94SO2CH3, xe2x80x94SO2NH2, xe2x80x94SO2NHC(O)CF3, xe2x80x94SO(NH)NH2 and xe2x80x94SO(NH)NHC(O)CF3;
each R2 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, amino, substituted amino, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halo, hydroxyl, nitro, thioalkoxy and substituted thioalkoxy;
R3 is a covalent bond linking the ligand to a linker; and
xcex1 is an integer from 0 to 3; and pharmaceutically-acceptable salts thereof.
Preferably, in the ligands of formula IA, ring A, together with the atoms to which it is attached, forms a cyclobut-2-en-1-one, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, 5(H)-furanone, benzene, pyridine, imidazopyridine, imidazothiazole or thiazolotriazole ring.
In the ligands of formula IB, ring B, together with the atoms to which it is attached, preferably forms a pyrazole ring.
Preferably, in the ligands of formula IC, ring C, together with the atoms to which it is attached, forms an imidazole ring.
In still another of its composition aspects, this invention provides a multibinding compound of formula II:
Lxe2x80x2xe2x80x94Xxe2x80x2xe2x80x94Lxe2x80x2xe2x80x83xe2x80x83II
wherein each Lxe2x80x2 is independently a ligand comprising a moiety capable of binding to cyclooxygenase-2 and Xxe2x80x2 is a linker; and pharmaceutically-acceptable salts thereof.
Preferably, in the multibinding compound of formula II, each ligand, Lxe2x80x2, is independently selected from the group consisting of:
(a) a compound of formula IIA: 
xe2x80x83wherein
R4 is selected from the group consisting of xe2x80x94SO2CH3, xe2x80x94SO2NH2, xe2x80x94SO2NHC(O)CF3, xe2x80x94SO(NH)NH2 and xe2x80x94SO(NH)NHC(O)CF3;
each R5 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, amino, substituted amino, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halo, hydroxyl, nitro, thioalkoxy and substituted thioalkoxy;
R6 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, cyano, fluoro and heteroaryl;
R7 is a covalent bond linking the ligand to a linker; and
b is an integer from 0 to 3;
(b) a compound of formula IIB: 
xe2x80x83wherein
R8 is selected from the group consisting of xe2x80x94SO2CH3, xe2x80x94SO2NH2, xe2x80x94SO2NHC(O)CF3, xe2x80x94SO(NH)NH2 and xe2x80x94SO(NH)NHC(O)CF3;
each R9 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, amino, substituted amino, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halo, hydroxyl, nitro, thioalkoxy and substituted thioalkoxy;
R10 is a covalent bond linking the ligand to a linker;
R11 is selected from the group consisting of hydrogen, alkyl, substituted alkyl and fluoro; and
c is an integer from 0 to 3; and
(c) a compound of formula IIC: 
xe2x80x83wherein
R12 is selected from the group consisting of xe2x80x94SO2CH3, xe2x80x94SO2NH2, xe2x80x94SO2NHC(O)CF3, xe2x80x94SO(NH)NH2 and xe2x80x94SO(NH)NHC(O)CF3;
each R13 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, amino, substituted amino, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halo, hydroxyl, nitro, thioalkoxy and substituted thioalkoxy;
R14 and R16 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl and fluoro;
R15 is a covalent bond linking the ligand to a linker; and
d is an integer from 0 to 3;
and pharmaceutically-acceptable salts thereof.
In a preferred embodiment, this invention is also directed to a multibinding compound of formula III: 
wherein
each R17 is independently selected from the group consisting of xe2x80x94SO2CH3, xe2x80x94SO2NH2, xe2x80x94SO2NHC(O)CF3, xe2x80x94SO(NH)NH2 and xe2x80x94SO(NH)NHC(O)CF3;
each R18 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, amino, substituted amino, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halo, hydroxyl, nitro, thioalkoxy and substituted thioalkoxy;
each R19 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, cyano, fluoro and heteroaryl;
e is an integer from 0 to 3; and
Xxe2x80x3 is a linker; and pharmaceutically-acceptable salts thereof.
In another preferred embodiment, this invention is directed to a multibinding compound of formula IV: 
wherein
each R20 is independently selected from the group consisting of xe2x80x94SO2CH3, xe2x80x94SO2NH2, xe2x80x94SO2NHC(O)CF3, xe2x80x94SO(NH)NH2 and xe2x80x94SO(NH)NHC(O)CF3;
each R21 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, amino, substituted amino, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halo, hydroxyl, nitro, thioalkoxy and substituted thioalkoxy;
f is an integer from 0 to 3; and
Xxe2x80x3 is a linker; and pharmaceutically-acceptable salts thereof.
In still another preferred embodiment, this invention is directed to a multibinding compound of formula V: 
wherein
each R22 is independently selected from the group consisting of xe2x80x94SO2CH3, xe2x80x94SO2NH2, xe2x80x94SO2NHC(O)CF3, xe2x80x94SO(NH)NH2 and xe2x80x94SO(NH)NHC(O)CF3;
each R23 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, amino, substituted amino, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halo, hydroxyl, nitro, thioalkoxy and substituted thioalkoxy;
each R24 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, cyano, fluoro and heteroaryl;
g is an integer from 0 to 3; and
Xxe2x80x3 is a linker; and pharmaceutically-acceptable salts thereof.
Preferably, in the above embodiments, each linker (i.e., X, Xxe2x80x2 or Xxe2x80x3) independently has the formula:
xe2x80x94Xaxe2x80x94Zxe2x80x94(Yaxe2x80x94Z)mxe2x80x94Ybxe2x80x94Zxe2x80x94Xaxe2x80x94
wherein
m is an integer of from 0 to 20;
Xa at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)NRxe2x80x94, xe2x80x94C(S), xe2x80x94C(S)Oxe2x80x94, xe2x80x94C(S)NRxe2x80x94 or a covalent bond where R is as defined below;
Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;
Ya and Yb at each separate occurrence are selected from the group consisting of xe2x80x94C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2C(O)xe2x80x94, xe2x80x94NRxe2x80x2C(O)NRxe2x80x2xe2x80x94, xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x95x90C(Xa)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94P(O)(ORxe2x80x2)xe2x80x94Oxe2x80x94, xe2x80x94S(O)nCRxe2x80x2Rxe2x80x3xe2x80x94, xe2x80x94S(O)nxe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94Sxe2x80x94Sxe2x80x94 and a covalent bond; where n is 0, 1 or 2; and R, Rxe2x80x2 and Rxe2x80x3 at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic.
In yet another of its composition aspects, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding to cyclooxygenase-2; and pharmaceutically-acceptable salts thereof.
This invention is also directed to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound of formula I, II, III, IV or V.
The multibinding compounds of this invention are effective inhibitors of the enzyme cyclooxygenase-2 (COX-2), an enzyme involved in the biosynthesis of prostaglandins associated with inflammation. Accordingly, in one of its method aspects, this invention provides a method for treating inflammation or an inflammation-related disorder in a patient, the method comprising administering to a patient having inflammation or an inflammation-related disorder a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding to cyclooxygenase-2; and pharmaceutically-acceptable salts thereof.
This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for cyclooxygenase-2. The diverse multimeric compound libraries provided by this invention are synthesized by combining a library of linkers with a library of ligands each having complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
Additionally, this invention is directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for cyclooxygenase-2. These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands for cyclooxygenase-2.
Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for cyclooxygenase-2, which method comprises:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in the library prepared in (c) above to identify multimeric ligand compounds possessing multibinding properties for cyclooxygenase-2.
In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for cyclooxygenase-2, which method comprises:
(a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in the library prepared in (c) above to identify multimeric ligand compounds possessing multibinding properties for cyclooxygenase-2.
Preferably, in these methods, the preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b).
Additionally, the multimeric ligand compounds comprising the multimeric ligand compound library are preferably dimeric. In one embodiment, the dimeric ligand compounds comprising the dimeric ligand compound library are heterodimeric. The heterodimeric ligand compound library is preferably prepared by sequential addition of a first and second ligand.
In a preferably embodiment of the above methods, prior to procedure (d), each member of the multimeric ligand compound library is isolated from the library. More preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).
In the above methods, the linker or linkers employed are preferably selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and/or polarizability and amphiphilic linkers. More preferably, the linkers comprise linkers of different chain length and/or having different complementary reactive groups. Still more preferably, the linkers are selected to have different linker lengths ranging from about 2 to 100 xc3x85.
The ligand or mixture of ligands employed in the above methods is preferably selected to have reactive functionality at different sites on said ligands. More preferably, the reactive functionality is selected from the group consisting of carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof wherein the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
In one preferred embodiment of the above methods, the multimeric ligand compound library comprises homomeric ligand compounds. In another preferred embodiment, the multimeric ligand compound library comprises heteromeric ligand compounds.
In one of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties. for cyclooxygenase-2, which library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties for cyclooxygenase-2, which library is prepared by the method comprising:
(a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In a preferred embodiment, the linker or linkers employed are preferably selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and/or polarizability and amphiphilic linkers. More preferably, the linkers comprise linkers of different chain length and/or having different complementary reactive groups. Still more preferably, the linkers are selected to have different linker lengths ranging from about 2 to 100 xc3x85.
In the above libraries, the ligand or mixture of ligands is preferably selected to have reactive functionality at different sites on said ligands. Preferably, the reactive functionality is selected from the group consisting of carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof wherein the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
In one embodiment, the multimeric ligand compound library comprises homomeric ligand compounds (i.e., each of the ligands is the same, although it may be attached at different points). In another embodiment, the multimeric ligand compound library comprises heteromeric ligand compounds (i.e., at least one of the ligands is different from the other ligands).
In another of its method aspects, this invention is directed to an iterative method for identifying multimeric ligand compounds possessing multibinding properties for cyclooxygenase-2, which method comprises:
(a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which target a receptor with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties for cyclooxygenase-2;
(c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties for cyclooxygenase-2;
(d) evaluating what molecular constraints imparted or are consistent with imparting multibinding properties to the multimeric compound or compounds found in the first iteration recited in (a)-(c) above;
(e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration;
(f) evaluating what molecular constraints imparted or are consistent with imparting enhanced multibinding properties to the multimeric compound or compounds found in the second collection or iteration recited in (e) above;
(g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.
Preferably, steps (e) and (f) are repeated from 2-50 times. More preferably, steps (e) and (f) are repeated from 5-50 times.
Preferably, the ligands employed in the above methods and library compositions are selected from ligands of formula IA-IC, more preferably, from ligands of formula IIA-IIC.